Although Wnt signaling is known to mediate multiple biological and pathological processes, its association with diabetic retinopathy (DR) has not been established. Here we show that retinal levels and nuclear translocation of ␤-catenin, a key effector in the canonical Wnt pathway, were increased in humans with DR and in three DR models. Retinal levels of lowdensity lipoprotein receptor-related proteins 5 and 6, coreceptors of Wnts , were also elevated in the DR models. The high glucose-induced activation of ␤-catenin was attenuated by aminoguanidine, suggesting that oxidative stress is a direct cause for the Wnt pathway activation in diabetes. Indeed, Dickkopf homolog 1, a specific inhibitor of the Wnt pathway, ameliorated retinal inflammation, vascular leakage, and retinal neovascularization in the DR models. Dickkopf homolog 1 also blocked the generation of reactive oxygen species induced by high glucose, suggesting that Wnt signaling contributes to the oxidative stress in diabetes. These observations indicate that the Wnt pathway plays a pathogenic role in DR and represents a novel therapeutic target. Diabetic retinopathy (DR), the leading cause of blindness in the working age population, represents a common concern in types 1 and 2 of diabetes mellitus (DM). 1 Accumulating evidence suggests that DR is a chronic inflammatory disorder.2 Retinal inflammation is believed to play a causative role in vascular leakage, which can lead to diabetic macular edema, and in retinal neovascularization (NV). It has been shown that levels of soluble intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 are significantly higher in the vitreous from patients with proliferative diabetic retinopathy than in nondiabetic vitreous. 3,4 Increased ICAM-1, vascular cell adhesion molecule-1, and e-selectin levels were found in the serum from patients with diabetic microangiopathy. [5][6][7] In diabetic animal models, increased retinal ICAM-1 expression is believed to be responsible for leukocyte adhesion or leukostasis and increased vascular permeability. Leukostasis is believed to contribute to capillary nonperfusion and local ischemia, which subsequently induces the overexpression of vascular endothelial growth factor (VEGF). 8 -11 Increased VEGF levels are responsible for the retinal vascular leakage and retinal NV.12,13 Recent studies have indicated that oxidative stress, induced by hyperglycemia, contributes to retinal inflammation in diabetes.14,15 However, the pathogenic mechanisms by which diabetes and oxidative stress induce inflammation are not certain at the present time.Wnts are a group of secreted, cysteine-rich glycoproteins, which bind to a coreceptor complex of frizzled (Fz) receptors and low-density lipoprotein receptor-related protein 5 or 6 (LRP5/6) and regulate expression of a number of target genes through an intracellular signaling pathway, namely the Wnt pathway. 16 In the absence of Wnt ligands, ␤-catenin, a down-stream effector of the canonical Wnt pathway, is phosphorylated by a pro...

Wnt signaling is known to regulate multiple processes including angiogenesis, inflammation, and fibrosis. Here, we identified a novel inhibitor of the Wnt pathway, pigment epithelium-derived factor (PEDF), a multifunctional serine proteinase inhibitor. Both overexpression of PEDF in transgenic mice and administration of PEDF protein attenuated Wnt signaling induced by retinal ischemia. Furthermore, PEDF knockdown by small interfering RNA (siRNA) and PEDF knockout in PEDF ؊/؊ mice induced activation of Wnt signaling. PEDF bound to LRP6, a Wnt coreceptor, with high affinity (K d [dissociation constant] of 3.7 nM) and blocked the Wnt signaling induced by Wnt ligand. The physical interaction of PEDF with LRP6 was confirmed by a coprecipitation assay, which showed that PEDF bound to LRP6 at the E1E2 domain. In addition, binding of PEDF to LRP6 blocked Wnt ligand-induced LRP6-Frizzled receptor dimerization, an essential step in Wnt signaling. These results suggest that PEDF is an endogenous antagonist of LRP6, and blocking Wnt signaling may represent a novel mechanism for its protective effects against diabetic retinopathy.

Activation of the canonical Wnt pathway is sufficient to induce retinal inflammation and oxidative stress and plays a pathogenic role in AMD and DR.

Aims/hypothesis The wingless-type MMTV integration site (WNT) pathway mediates multiple physiological and pathological processes, such as inflammation, angiogenesis and fibrosis. The aim of this study was to investigate whether canonical WNT signalling plays a role in the pathogenesis of diabetic nephropathy. Methods Expression of WNT ligands and frizzled receptors in the canonical WNT pathway in the kidney was compared at the mRNA level using real-time RT-PCR between Akita mice, streptozotocin-induced diabetic rats and db/db mice and their respective non-diabetic controls. Renal function was evaluated by measuring the urine albumin excretion. Human renal proximal tubular epithelial cells were treated with high-glucose medium and 4-hydroxynonenal (HNE). Levels of β-catenin, connective tissue growth factor and fibronectin were determined by western blot analysis. Results Some of the WNT ligands and frizzled receptors showed increased mRNA levels in the kidneys of Akita mice, streptozotocin-induced diabetic rats and db/db mice compared with their non-diabetic controls. Renal levels of β-catenin and WNT proteins were upregulated in these diabetic models. Lowering the blood glucose levels by insulin attenuated the activation of WNT signalling in the kidneys of Akita mice. In cultured human renal proximal tubular epithelial cells, both high glucose and HNE activated WNT signalling. Inhibition of WNT signalling with a monoclonal antibody blocking LDL-receptor-related protein 6 ameliorated renal inflammation and fibrosis and reduced proteinuria in Akita mice. Conclusions/interpretation The WNT pathway is activated in the kidneys of models of both type 1 and 2 diabetes. Dysregulation of the WNT pathway in diabetes represents a new pathogenic mechanism of diabetic nephropathy and renders a new therapeutic target.

Long non-coding RNAs (lncRNAs) have previously been implicated in human disease states, especially cancer. Although the aberrant expression of lncRNAs has been observed in cancer, the biological functions and molecular mechanisms underlying aberrantly expressed lncRNAs in hepatocellular carcinoma (HCC) have not been widely established. In the present study, we investigated a novel lncRNA, termed URHC (up-regulated in hepatocellular carcinoma), and evaluated its role in the progression of HCC. Expression profiling using a lncRNA microarray revealed that URHC was highly expressed in 3 HCC cell lines compared to normal hepatocytes. Quantitative real-time polymerase chain reaction (qRT-PCR) analyses confirmed that URHC expression was increased in hepatoma cells and HCC tissues. Moreover, using qRT-PCR, we confirmed that URHC expression was up-regulated in 30 HCC cases (57.7%) and that its higher expression was correlated with poor overall survival. We further demonstrated that URHC inhibition reduced cell proliferation and promoted apoptosis. We hypothesize that URHC may function by regulating the sterile alpha motif and leucine zipper containing kinase AZK (ZAK) gene, which is located near URHC on the same chromosome. We found that ZAK mRNA levels were down-regulated in HCC tissues and the expression levels of ZAK were negatively correlated with those of URHC in the above HCC tissues. Next, we confirmed that URHC down-regulated ZAK, which is involved in URHC-mediated cell proliferation and apoptosis. Furthermore, ERK/MAPK pathway inactivation partially accounted for URHC-ZAK-induced cell growth and apoptosis. Thus, we concluded that high URHC expression can promote cell proliferation and inhibit apoptosis by repressing ZAK expression through inactivation of the ERK/MAPK pathway. These findings may provide a novel mechanism and therapeutic targets for the treatment of HCC.

The Wnt pathway regulates multiple biological and pathological processes including angiogenesis and inflammation. Here we identified a unique inhibitor of the Wnt pathway, SERPINA3K, a serine proteinase inhibitor with anti-inflammatory and angiogenic activities. SERPINA3K blocked the Wnt pathway activation induced by a Wnt ligand and by diabetes. Coprecipitation and ligand binding assay showed that SERPINA3K binds to low-density lipoprotein receptor-like protein 6 (LRP6) with a K d of 10 nM, in the range of its physiological concentration in the retina. Under the same conditions, SERPINA3K did not bind to the frizzled (Fz) receptor or lowdensity lipoprotein receptor. Further, SERPINA3K bound to LRP6 at the extracellular domain and blocked its dimerization with the Fz receptor induced by a Wnt ligand. The antagonizing activity of SER-PINA3K to LRP6 was further confirmed by Xenopus axis duplication assay. These results suggest that SERPINA3K is a high-affinity, endogenous antagonist of LRP6. The blockade of Wnt signaling may represent a unifying mechanism for the anti-inflammatory and antiangiogenic effects of SERPINA3K.angiogenesis | β-catenin | lipoprotein receptor-related protein 6 | vascular endothelial growth factor

Diabetic foot ulcer (DFU) caused by impaired wound healing is a common vascular complication of diabetes. The current study revealed that plasma levels of pigment epithelium-derived factor (PEDF) were elevated in type 2 diabetic patients with DFU and in db/db mice. To test whether elevated PEDF levels contribute to skin wound-healing delay in diabetes, endogenous PEDF was neutralized with an anti-PEDF antibody in db/db mice. Our results showed that neutralization of PEDF accelerated wound healing, increased angiogenesis in the wound skin, and improved the functions and numbers of endothelial progenitor cells (EPCs) in the diabetic mice. Further, PEDF-deficient mice showed higher baseline blood flow in the skin, higher density of cutaneous microvessels, increased skin thickness, improved numbers and functions of circulating EPCs, and accelerated wound healing compared with wild-type mice. Overexpression of PEDF suppressed the Wnt signaling pathway in the wound skin. Lithium chlorideinduced Wnt signaling activation downstream of the PEDF interaction site attenuated the inhibitory effect of PEDF on EPCs and rescued the wound-healing deficiency in diabetic mice. Taken together, these results suggest that elevated circulating PEDF levels contribute to impaired wound healing in the process of angiogenesis and vasculogenesis through the inhibition of Wnt/b-catenin signaling.

Aims/hypothesisOur recent studies suggest that activation of the wingless-type MMTV integration site (WNT) pathway plays pathogenic roles in diabetic retinopathy and age-related macular degeneration. Here we investigated the causative role of oxidative stress in retinal WNT pathway activation in an experimental model of diabetes.MethodsCultured retinal pigment epithelial cells and retinal capillary endothelial cells were treated with a lipid peroxidation product, 4-hydroxynonenal (HNE), and an antioxidant, N-acetyl-cysteine (NAC). In vivo, rats with streptozotocin-induced diabetes were treated by NAC for 8 weeks. Activation of the canonical WNT pathway was measured by TOPFLASH assay and by western blot analysis of WNT pathway components and a WNT target gene, Ctgf. Oxidative stress in the retina was evaluated by immunostaining of HNE and 3-nitrotyrosine.ResultsLevels of phosphorylated and total LDL receptor-related protein (LRP)6, and cytosolic β-catenin, as well as transcriptional activity of T cell factor (TCF)/β-catenin were significantly increased by HNE. The production of connective tissue growth factor (CTGF) was also upregulated by HNE. NAC blocked the WNT pathway activation induced by HNE. Furthermore, LRP6 stability was increased by HNE and decreased by NAC. Retinal levels of HNE and 3-nitrotyrosine were significantly increased in diabetic rats, compared with those in non-diabetic rats. In the same diabetic rat retinas, levels of LRP6, cytosolic β-catenin and CTGF were significantly increased. NAC treatment reduced HNE and 3-nitrotyrosine levels and attenuated the upregulation of LRP6, β-catenin and CTGF in diabetic rat retina.Conclusions/interpretationLipid peroxidation products activate the canonical WNT pathway through oxidative stress, which plays an important role in the development of retinal diseases.